1. Field of the Invention
This invention relates to the formation of agarose beads, preferably incorporating biological materials such as mammalian cells or cellular components (organelles) in which the usefulness of the biological materials in laboratory and industrial processes is maintained after the formation process.
2. Statement of Related Art
There is increasing utilization of biological materials incorporated into algal polysaccharide gels in biotechnology. While the incorporation of biological materials in alginates and carrageenan, and the subsequent formation of beads are known in the art, the successful incorporation of biological materials as defined herein in agarose maintained at bioprocess grade without extraneous contamination has not previously been accomplished.
As used herein, the term "biological material(s)" includes material that is capable of self-replication either directly or indirectly. Representative examples include: bacteria; fungi including yeast; algae; protozoa; mammalian, especially eukaryotic cells; animal cell lines; hybridomas; plasmids; viruses; plant tissue cells; lichens; and seeds. "Biological material" also includes all cell segments and other non-living material of biological origin such as: proteins; enzymes and enzyme systems; vectors; and all organelles; as well as indirect replicating materials including: phages; plasmids; symbiants; and replication defective cells. All "biological materials" as defined in this invention are limited in that they are at least partially labile at 40.degree. C. or above, especially at 37.degree. C. or above. Eukaryotic cells are the preferred biological material in this invention, eukaryotic mammalian cells being particularly preferred.
Agarose has various desirable features over other gels, for example:
1. Agarose is clearer than other natural gelling media such as those requiring cations (e.g. alginates, carrageenan). As a result cells incorporated in agarose are more easily visualized, permitting easier differentiation.
2. Agarose does not require polymerization, as does hydroxyethylmethacrylate (HEMA) and polyacrylamide gel (PAG) both of which are used for cell culturing.
3 The presence of cations (notably calcium) used for formation of other types of gels influences cell proteins and cell metabolic functions and therefore is undesirable. Agarose does not contain such cations, since they are not utilized in its gelation.
4. Cells can be recovered from an agarose gel by a brief freeze/thaw process, which causes gel syneresis, without using the chelators normally required for alginate gels. Such chelators can adversely affect cell viability.
5. Agarose is a selective medium for transformed cells (e.g. hybridomas, certain tumor cells, and the like). It is a prerequisite for a cell line to be considered "transformed", that it be grown successfully, i.e., that it becomes "immortal".
There are, however, considerable difficulties in the formation of "beads" from agarose. The term "beads" is used herein in its normal biochemical laboratory sense, that is, as referring to small, discrete gel particles.
Agarose will only gel when cooled below its gelling temperature. It will not gel in response to cations such as calcium, which are effective for gelling alginate and some carrageenans. As a result, the earliest art describing the formation of agarose beads (for chromatographic application) used warm, non-aqueous solvents in which the agarose was emulsified before gel formation by cooling. There are numerous references to the formation of agarose beads, including: Hjerten, S. Biochim. Biophys. Acta 1964, 79:393-398; and Bengtsson et al., S. Biochim. Biophys. Acta 1964. 79:399, which teaches spraying the agarose into "ice-cold" ether. The use of organic solvents in agarose bead formation is still being utilized. The utilization of organic solvents in the formation of agarose beads renders such beads unsuitable for the incorporation of biological materials, since most such solvents are toxic to the cells at the concentrations in which they are used, and cannot satisfactorily be removed from the beads without damaging the incorporated material.
Another method for agarose bead formation, as disclosed in U.S. Pat. No. 4,647,536 is dropping an agarose emulsion into a cooled oil. Such a method is also disclosed in "Methods in Enzymology" Vol. 135 Part B, p. 401, Academic Press, 1987, which suggests the use of paraffin oil but cautions that the oil must be washed five times before use in order to remove any cytotoxins present in the oil. Therefore, after formation of the agarose beads using such procedure, they must be repeatedly washed and screened to free them of residual oil that would affect cell growth, enzyme production, etc. since, the presence of oil and/or solvent contamination renders agarose beads undesirable for biological material and particularly cell and cellular fragment entrapment.
U.S. Pat. No. 4,647,536 describes a general method for immobilization of animal and other cells in agarose and other reagents, and is incorporated herein by reference for its disclosure of such cells.
Nilsson et al., in "Preparation of Immobilized Animal Cells" [FEBS, 118:145-150 (1980)], discloses the formation of agarose beads incorporating a cell suspension by dissolving the agarose, admixing the cell suspension, and then pouring the mixture over a polytetrafluoroethylene plate which is "tightly covered with 3 mm holes". Another plate was used as a support and the two were held together by clamps. Nilsson et al., disclose that in the molding process, the agarose solidified into cylindrical beads. Nilsson et al., do not disclose any reason for using polytetrafluoroethylene as the mold form, and it can reasonably be assumed that it was used to facilitate extraction.
U.S. Pat. No. 4,722,898-Errede et al., discloses the immobilization of biological cells in a polytetrafluoroethylene matrix per se, and contrasts such material with the use of gelled polymers at column 2 line 47. The patent discloses the admixture of nutrient ingredients with the polytetrafluoroethylene matrix, including synthetic organic compounds derived from the polysaccharide dextran. There is, however, no disclosure of the use of agarose. A number of cells and microorganisms suitable for entrapment by the process of the subject invention are disclosed and, to the extent they are labile at 40.degree. C. or above, that disclosure in U.S. Pat. No. 4,722,898 is incorporated herein by reference.
U.S. Pat. No. 4,778,749 - Vasington, et al., discloses the formation of hydrophilic gel beads incorporating mammalian cells and hybridomas. The only gels for which there are examples or enabling disclosure are alginates, specifically sodium alginates, which are gelled by the addition of a calcium salt. There is a statement at column 3, lines 39-42 that "other hydrophilic materials such as agarose, agar, carrageenan, xanthan gum, polyacrylamides, poly HEMA, and others known in the art can be used to advantage in particular environments". There is no further disclosure enabling the use of agarose, distinguishing agarose from the other listed materials, or recognizing that forming beads from agarose presents particular problems. This patent is incorporated by reference for its disclosures of biological materials capable of incorporation (entrapment) and for the formation of droplets of hydrogel/biological material aqueous solution, but not for the method of cooling (gelling) the droplets, which are contrary to the teachings of the present invention.
Of the various algal polysaccharides, whose gelation is not cation dependent, only agarose (and agar) will remain liquid at a temperature below 40.degree. C., preferably 37.degree. C., the temperature above which the viability of the biological materials, especially mammalian cells, becomes impaired. For this reason, agarose is a very desirable medium for the incorporation of living cells and viable cell fragments and subsequent bead formation. It is much preferred over agar because of its higher purity.
However, when such cellular material is incorporated in agarose and bead formation is attempted by the known bead formation method of dropping particles into cold (0.degree.-25.degree. C.) water, the result is totally unsatisfactory. Liquefied agarose dropped into cold water results in the formation of particles having unknown gel concentration, gradients, irregular surfaces and erratic geometry, any one of which is very undesirable.
The problems presented by prior art processes for the formation of agarose beads incorporating biological materials are, in summary:
(A) using gels which require maintenance at a temperature of over 40.degree. C., especially over 37.degree. C. to remain in a liquid state (i.e., which gel at about 40.degree. C.), which temperature at least partially destroys the incorporated biological materials as defined herein; PA1 (B) the presence of organic solvents and/or of oil contaminants as a result of the bead formation process; and/or PA1 (C) the inability to produce beads having regular surfaces and geometry, known gel concentration, and little or no concentration gradient. [Bead irregularity (e.g. the presence of appendages such as tails, skirts, etc.) is a problem because such appendages break-off and clog downstream filters, as well as loosen some incorporated cells which themselves can become a contaminant.] PA1 A) forming an aqueous solution comprising an agarose which gels at a temperature of about 40.degree. C. or less; PA1 B) cooling the agarose solution to 40.degree. C. or less while maintaining it in liquid form; PA1 C) admixing an aqueous broth containing biological material which is at least partially labile at above 40.degree. C. with the gel solution, to form a liquid agarose/broth mixture; PA1 D) forming bead-size portions of the liquid agarose/broth mixture; PA1 E) contacting said portions with a cooled atmosphere, gas, and/or smooth hydrophobic surface and maintaining such contact until said portions gel into formed agarose beads containing biological material; and PA1 F) collecting the formed beads.